Summary of Proposal: This proposal supports a 5-year mentored career development award investigating the tissue-based immune response through the lens of a recent phase 1 immunotherapeutic vaccine trial with the long-term goal of contributing to the development of an effective herpes simplex virus-2 (HSV-2) vaccine and improving global health.. This trial involved three sequential doses of an immunotherapeutic vaccine, HSV529, in individuals with symptomatic HSV-2 infection. T-cell receptor (TCR) repertoire analysis was performed of T cells in small biopsies of the area of HSV-2 reactivation in comparison to T cells from biopsies of uninvolved skin and HSV-2 reactive CD4+ T cells from peripheral blood to research two main lines of inquiry. The first is the interrelatedness, over time, of the TCR repertoire of the genital skin at HSV-2 reactivation sites, the arm, and HSV-2 specific CD4+ T cells over time; whether T cells detected in genital tissue are detectable in blood with standard sampling techniques. The second is how these relationships and dynamics change over the course of an immunotherapeutic vaccination. Preliminary findings suggest that some T cells increased in number after vaccination in everyone, with very little overlap between the skin and the blood. The central hypothesis is that T cells that respond to a vaccine targeting HSV-2 are likely to be HSV-2-specific, and that T cells seen in tissue at high copy number over a long period of time are likely to be tissue residents. The objective of this proposal is to learn more about these resident T cells, which are otherwise difficult to study. The rationale for this is that vaccine development for HSV-2 has thus far not been successful, and that because there is so little overlap between the skin and the blood, that looking directly at the tissue may help identify a viral antigen or a way to target skin-based T cells to improve vaccine efficacy. Research plan: AIM 1a will identify the specificity of T cells of that were seen most frequently in the region of HSV-2 reactivation, focusing on T cells that expanded in number after immunotherapeutic vaccination, as these are the most likely to be herpes-specific, tissue-resident cells. AIM 1b focuses on establishing the spatial location of these cells in small sections of biopsies using fluorescent molecular tags to visualize their arrangement in skin. AIM 2 focuses on learning more about these T cells by using transcriptional profiling, a technique that separates each of the T cells in a biopsy and looks at them one by one to uncover the cellular processes that define the activity of each cell. We will incorporate information from TCR repertoire analysis to interpret the results of transcriptional profiling more effectively and accurately. This approach uses small skin samples in the most comprehensive manner available to describe the fight between HSV-2 and the human immune system. By conducting this research with an extremely experienced and uniquely skilled group of mentors in the rich educational environment of the FHCRC and UW, Dr. Ford will become an effective and productive lead investigator.